CD19 Regulates Skin and Lung Fibrosis via Toll-Like Receptor Signaling in a Model of Bleomycin-Induced Scleroderma
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.071049
ISSN1525-2191
AutoresAyumi Yoshizaki, Yohei Iwata, Kazuhiro Komura, Fumihide Ogawa, Toshihide Hara, Eiji Muroi, Motoi Takenaka, Kazuhiro Shimizu, Minoru Hasegawa, Manabu Fujimoto, Thomas F. Tedder, Shinichi Sato,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoMice subcutaneously injected with bleomycin, in an experimental model of human systemic sclerosis, develop cutaneous and lung fibrosis with autoantibody production. CD19 is a general “rheostat” that defines signaling thresholds critical for humoral immune responses, autoimmunity, and cytokine production. To determine the role of CD19 in the bleomycin-induced systemic sclerosis model, we investigated the development of fibrosis and autoimmunity in CD19-deficient mice. Bleomycin-treated wild-type mice exhibited dermal and lung fibrosis, hyper-γ-globulinemia, autoantibody production, and enhanced serum and skin expression of various cytokines, including fibrogenic interleukin-4, interleukin-6, and transforming growth factor-β1, all of which were inhibited by CD19 deficiency. Bleomycin treatment enhanced hyaluronan production in the skin, lung, and sera. Addition of hyaluronan, an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, stimulated B cells to produce various cytokines, primarily through TLR4; CD19 deficiency suppressed this stimulation. These results suggest that bleomycin induces fibrosis by enhancing hyaluronan production, which activates B cells to produce fibrogenic cytokines mainly via TLR4 and induce autoantibody production, and that CD19 deficiency suppresses fibrosis and autoantibody production by inhibiting TLR4 signals. Mice subcutaneously injected with bleomycin, in an experimental model of human systemic sclerosis, develop cutaneous and lung fibrosis with autoantibody production. CD19 is a general “rheostat” that defines signaling thresholds critical for humoral immune responses, autoimmunity, and cytokine production. To determine the role of CD19 in the bleomycin-induced systemic sclerosis model, we investigated the development of fibrosis and autoimmunity in CD19-deficient mice. Bleomycin-treated wild-type mice exhibited dermal and lung fibrosis, hyper-γ-globulinemia, autoantibody production, and enhanced serum and skin expression of various cytokines, including fibrogenic interleukin-4, interleukin-6, and transforming growth factor-β1, all of which were inhibited by CD19 deficiency. Bleomycin treatment enhanced hyaluronan production in the skin, lung, and sera. Addition of hyaluronan, an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, stimulated B cells to produce various cytokines, primarily through TLR4; CD19 deficiency suppressed this stimulation. These results suggest that bleomycin induces fibrosis by enhancing hyaluronan production, which activates B cells to produce fibrogenic cytokines mainly via TLR4 and induce autoantibody production, and that CD19 deficiency suppresses fibrosis and autoantibody production by inhibiting TLR4 signals. Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and other visceral organs with an autoimmune background.1LeRoy EC Krieg T Black C Medsger TAJ Fleischmajer R Rowell N Jablonska S Wollheim F Scleroderma (systemic sclerosis): classification, subsets, and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar The presence of autoantibodies is a central feature of SSc, because antinuclear antibodies (Abs) are detected in >90% of patients.2Okano Y Antinuclear antibody in systemic sclerosis (scleroderma).Rheum Dis Clin North Am. 1996; 22: 709-735Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar SSc patients have autoantibodies that react to various intracellular components, such as DNA topoisomerase I (topo I), centromeric protein B (CENP B), U1-ribonucleoprotein (RNP), and histones.2Okano Y Antinuclear antibody in systemic sclerosis (scleroderma).Rheum Dis Clin North Am. 1996; 22: 709-735Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Furthermore, abnormal activation of immune cells, including T lymphocytes, B lymphocytes, natural killer cells, and macrophages, has been identified in SSc.3Sato S Fujimoto M Hasegawa M Takehara K Altered blood B lymphocyte homeostasis in systemic sclerosis: expanded naive B cells and diminished but activated memory B cells.Arthritis Rheum. 2004; 50: 1918-1927Crossref PubMed Scopus (254) Google Scholar, 4Gudbjornsson B Hallgren R Nettelbladt O Gustafsson R Mattsson A af Geijerstam E Totterman TH Phenotypic and functional activation of alveolar macrophages. T lymphocytes and NK cells in patients with systemic sclerosis and primary Sjogren's syndrome.Ann Rheum Dis. 1994; 53: 574-579Crossref PubMed Scopus (39) Google Scholar, 5Kalogerou A Gelou E Mountantonakis S Settas L Zafiriou E Sakkas L Early T cell activation in the skin from patients with systemic sclerosis.Ann Rheum Dis. 2005; 64: 1233-1235Crossref PubMed Scopus (102) Google Scholar A recent study has shown that skin and lung fibrosis is ameliorated by treatment with cyclophosphamide, an immunosuppressive agent, indicating that immune activation leads to fibrosis through the stimulation of collagen production by fibroblasts.6Tashkin DP Elashoff R Clements PJ Goldin J Roth MD Furst DE Arriola E Silver R Strange C Bolster M Seibold JR Riley DJ Hsu VM Varga J Schraufnagel DE Theodore A Simms R Wise R Wigley F White B Steen V Read C Mayes M Parsley E Mubarak K Connolly MK Golden J Olman M Fessler B Rothfield N Metersky M Cyclophosphamide versus placebo in scleroderma lung disease.N Engl J Med. 2006; 354: 2655-2666Crossref PubMed Scopus (1280) Google Scholar Indeed, SSc patients exhibit elevated serum levels of various cytokines, especially fibrogenic Th2 cytokines, such as interleukin (IL)-4, IL-6, IL-10, some Th1 cytokines, such as IL-2, tumor necrosis factor (TNF)-α, and IL-12, a transforming growth factor (TGF)-β1, a major fibrogenic growth factor. B-cell signaling thresholds are regulated by response regulators that augment or diminish B-cell signals during responses to self and foreign antigens.7Tedder TF Poe JC Fujimoto M Haas KM Sato S The CD19-CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system.Curr Dir Autoimmun. 2005; 8: 55-90Crossref PubMed Google Scholar Abnormal regulation of the response regulator function and expression may result in autoantibody production. Among these response regulators, CD19, which is a critical cell-surface signal transduction molecule of B cells, is a most potent positive regulator.7Tedder TF Poe JC Fujimoto M Haas KM Sato S The CD19-CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system.Curr Dir Autoimmun. 2005; 8: 55-90Crossref PubMed Google Scholar Transgenic mice that overexpress CD19 by approximately threefold lose tolerance and generate autoantibodies spontaneously.8Sato S Ono N Steeber DA Pisetsky DS Tedder TF CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.J Immunol. 1996; 157: 4371-4378PubMed Google Scholar, 9Inaoki M Sato S Weintraub BC Goodnow CC Tedder TF CD19-regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes.J Exp Med. 1997; 186: 1923-1931Crossref PubMed Scopus (162) Google Scholar Human SSc patients exhibit a 20% increase in CD19 expression, which is associated with −499G>T allele in the CD19 promoter.10Sato S Hasegawa M Fujimoto M Tedder TF Takehara K Quantitative genetic variation in CD19 expression correlates with autoimmunity.J Immunol. 2000; 165: 6635-6643PubMed Google Scholar, 11Tsuchiya N Kuroki K Fujimoto M Murakami Y Tedder TF Tokunaga K Takehara K Sato S Association of a functional CD19 polymorphism with susceptibility to systemic sclerosis.Arthritis Rheum. 2004; 50: 4002-4007Crossref PubMed Scopus (82) Google Scholar This CD19 overexpression may be related to autoantibody production and hyper-γ-globulinemia in human SSc, because mice that overexpress CD19 to a similar extent as human SSc have hyper-γ-globulinemia and elevated levels of various autoantibodies, including SSc-specific anti-topo I Ab.12Sato S Fujimoto M Hasegawa M Takehara K Tedder TF Altered B lymphocyte function induces systemic autoimmunity in systemic sclerosis.Mol Immunol. 2004; 41: 1123-1133Crossref PubMed Scopus (120) Google Scholar Furthermore, SSc patients have intrinsic B-cell abnormalities characterized by chronic hyperreactivity of memory B cells.3Sato S Fujimoto M Hasegawa M Takehara K Altered blood B lymphocyte homeostasis in systemic sclerosis: expanded naive B cells and diminished but activated memory B cells.Arthritis Rheum. 2004; 50: 1918-1927Crossref PubMed Scopus (254) Google Scholar In addition, the production of B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell stimulatory molecule, is up-regulated with an enhanced ability of SSc B cells to produce IgG and IL-6 by BAFF stimulation.13Matsushita T Hasegawa M Yanaba K Kodera M Takehara K Sato S Elevated serum BAFF levels in patients with systemic sclerosis: enhanced BAFF signaling in systemic sclerosis B lymphocytes.Arthritis Rheum. 2006; 54: 192-201Crossref PubMed Scopus (221) Google Scholar Thus, intrinsic B-cell abnormalities may play a role in the systemic autoimmunity of SSc. The loss of CD19 expression in a tight-skin (TSK) mouse, a genetic, spontaneous model of SSc, results in the inhibition of chronic B-cell hyperreactivity and in the elimination of autoantibody production, which is associated with improvement of skin fibrosis and a parallel decrease in IL-6 production by B cells.14Saito E Fujimoto M Hasegawa M Komura K Hamaguchi Y Kaburagi Y Nagaoka T Takehara K Tedder TF Sato S CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight-skin mouse.J Clin Invest. 2002; 109: 1453-1462Crossref PubMed Scopus (231) Google Scholar Furthermore, B-cell depletion by anti-CD20 Ab or treatment with BAFF antagonists improves skin fibrosis in TSK mice.15Matsushita T Fujimoto M Hasegawa M Matsushita Y Komura K Ogawa F Watanabe R Takehara K Sato S BAFF antagonist attenuates the development of skin fibrosis in tight-skin mice.J Invest Dermatol. 2007; 127: 2772-2780PubMed Scopus (66) Google Scholar, 16Hasegawa M Hamaguchi Y Yanaba K Bouaziz JD Uchida J Fujimoto M Matsushita T Matsushita Y Horikawa M Komura K Takehara K Sato S Tedder TF B-lymphocyte depletion reduces skin fibrosis and autoimmunity in the tight-skin mouse model for systemic sclerosis.Am J Pathol. 2006; 169: 954-966Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar These findings suggest that B cells play a critical role in the development of fibrosis as well as autoantibody production in TSK mice. However, this hypothesis has not been proven, because there are important differences in SSc between the TSK mouse model and humans. First, skin fibrosis in TSK mice occurs in the subcutaneous loose connective tissue layer, which does not exist in humans; in contrast, fibrosis in human SSc occurs in the dermis. Second, inflammation of the dermis, which regulates skin fibrosis by producing cytokines in human SSc, is very modest in TSK mice. Third, TSK mice exhibit lung emphysema, whereas human SSc is associated with lung fibrosis. Finally, because SSc occurs in only 1.6% of families with SSc,17Arnett FC Cho M Chatterjee S Aguilar MB Reveille JD Mayes MD Familial occurrence frequencies and relative risks for systemic sclerosis (scleroderma) in three United States cohorts.Arthritis Rheum. 2001; 44: 1359-1362Crossref PubMed Scopus (228) Google Scholar human SSc is not a genetic disorder as it is in TSK mice. Recently, Yamamoto and colleagues18Yamamoto T Takagawa S Katayama I Yamazaki K Hamazaki Y Shinkai H Nishioka K Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma.J Invest Dermatol. 1999; 112: 456-462Crossref PubMed Scopus (339) Google Scholar, 19Yamamoto T The bleomycin-induced scleroderma model: what have we learned for scleroderma pathogenesis?.Arch Dermatol Res. 2006; 297: 333-344Crossref PubMed Scopus (76) Google Scholar established a new mouse model of SSc using bleomycin (BLM) treatment: the subcutaneous injection of BLM induces fibrosis in the dermis and lung, autoantibody production, and dermal inflammatory infiltration, which more closely mimics the features of human SSc than that of TSK mice. However, the contribution of B cells and CD19 to disease manifestations and autoimmunity, and the mechanisms underlying B-cell activation by BLM, remain unknown in the BLM-induced SSc model. In this study, we investigated the role of CD19 in the development of autoimmunity and fibrosis induced by BLM using CD19-deficient (CD19−/−) mice. The results of this study indicate that CD19 regulates fibrogenic cytokine production by B cells mainly through Toll-like receptor (TLR) 4 signaling, which was activated by hyaluronan, an endogenous TLR4 ligand that is up-regulated in the dermis and lung by BLM treatment: CD19 thus controls skin and lung fibrosis, hyper-γ-globulinemia, and autoantibody production induced by BLM treatment. CD19−/− (C57BL/6 × 129) mice were generated as described20Engel P Zhou L-J Ord DC Sato S Koller B Tedder TF Abnormal B lymphocyte development, activation and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (490) Google Scholar and backcrossed 7 to 12 generations onto the C57BL/6 background before use in this study. Lack of cell surface CD19 expression was verified by two-color immunofluorescence staining with flow cytometric analysis. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. The mice used in these experiments were 6 weeks of age. All studies and procedures were approved by the Committee on Animal Experimentation of Nagasaki University Graduate School of Medical Science. BLM (Nippon Kayaku, Tokyo, Japan) was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml and sterilized by filtration. BLM or PBS (300 μg) was injected subcutaneously into the shaved backs of the mice daily for 4 weeks with a 27-gauge needle, as described previously.18Yamamoto T Takagawa S Katayama I Yamazaki K Hamazaki Y Shinkai H Nishioka K Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma.J Invest Dermatol. 1999; 112: 456-462Crossref PubMed Scopus (339) Google Scholar Morphological characteristics of skin sections were compared between CD19−/− and wild-type (WT) mice treated with either BLM or PBS under a light microscope. All skin sections were taken from the para-midline, lower back region (the same anatomical site, to minimize regional variations in thickness) as full-thickness sections extending down to the body wall musculature. Tissues were fixed in 10% formaldehyde solution for 24 hours and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E). Dermal thickness, defined as the thickness of skin from the top of the granular layer to the junction between the dermis and subcutaneous fat, was examined. Ten random measurements were taken per section. All of the sections were examined independently by two investigators in a blinded manner. The skin from male mice was generally thicker than that from female mice despite the BLM or PBS treatment (data not shown). Because similar results were obtained when male or female mice were analyzed separately, only data from female mice were presented for skin thickness in this study. Mast cells were identified by toluidine blue staining. Cells containing metachromatic granules were counted in 10 random grids under high-magnification (×400) power fields of a light microscope. Frozen tissue sections of skin biopsies were acetone-fixed and then incubated with 10% normal rabbit serum in PBS (10 minutes, 37°C) to block nonspecific staining. Sections were then incubated with rat monoclonal Ab (mAb) specific for macrophages (F4/80; Serotec, Oxford, UK), B220 (BD PharMingen, San Diego, CA), CD4 (clone RM4-5, BD PharMingen), and CD8 (clone 53-6.7, BD PharMingen). Rat IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 minutes, 37°C) with a biotinylated rabbit anti-rat IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and horseradish peroxidase-conjugated avidin-biotin complexes (Vectastain ABC kit, Vector Laboratories). Sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and then counterstained with methyl green. Stained cells were counted in 10 random grids under high-magnification (×400) power fields of a light microscope. Each section was examined independently by two investigators in a blinded manner. Lungs were excised after 4 weeks of treatment with BLM or PBS, processed as previously described,21Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1092) Google Scholar and stained by H&E and van Gieson to detect collagen. The severity of fibrosis was semiquantitatively assessed according to Ashcroft and colleagues.21Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1092) Google Scholar Briefly, the lung fibrosis was graded on a scale of 0 to 8 by examining randomly chosen fields of the left middle lobe at a magnification of ×100. The grading criteria were as follows: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; and grade 8, total fibrous obliteration of fields. Grades 2, 4, and 6 were used as intermediate pictures between the aforementioned criteria. All of the sections were scored independently by two investigators in a blinded manner. Formalin-fixed and paraffin-embedded tissues were cut into sections of 4 μm in thickness, deparaffinized in xylene, and rehydrated in PBS. Deparaffinized sections were preincubated with 1% H2O2 for 5 minutes to block tissue peroxidase activity. The sections were then incubated with bovine serum albumin in PBS for 30 minutes at 37°C, followed by overnight incubation at 4°C with 3 mg/ml of biotinylated hyaluronic acid-binding protein (Sigma-Aldrich, St. Louis, MO). After washing with PBS, the slides were incubated with streptavidin-horseradish peroxidase (BD PharMingen) for 1 hour, and the reaction products were visualized using diaminobenzidine with methyl green as counterstaining. The specificity of the staining was confirmed by preincubating the sections with Streptomyces-derived hyaluronidase to remove hyaluronan from the tissue. Sera were obtained by a cardiac puncture after 4 weeks of treatment with BLM or PBS and were stored at −80°C. Serum levels of IL-4, IL-6, IL-10, interferon (IFN)-γ, TGF-β1, TNF-α, and macrophage inflammatory protein (MIP)-2 were assessed using specific ELISA kits (IL-4, IL-6, IL-10, IFN-γ, TGF-β1, and TNF-α: Biosource International, Camarillo, CA; and MIP-2: Peprotech, London, UK). The amount of hyaluronan in the serum was quantified using an ELISA kit (Echelon Biosciences, Salt Lake City, UT). Antinuclear Abs were assessed by indirect immunofluorescence staining using sera diluted 1:50 and HEp-2 substrate cells (Medical & Biological Laboratories, Nagoya, Japan) as described.10Sato S Hasegawa M Fujimoto M Tedder TF Takehara K Quantitative genetic variation in CD19 expression correlates with autoimmunity.J Immunol. 2000; 165: 6635-6643PubMed Google Scholar Antinuclear Abs were detected using fluorescein isothiocyanate-conjugated F(ab′)2 fragments specific for mouse IgG + IgM + IgA (Southern Biotechnology Associates). The specific ELISA kits were used to measure anti-topo I (Medical & Biological Laboratories), anti-CENP B (Funakoshi, Tokyo, Japan), anti-U1-RNP (Medical & Biological Laboratories), anti-histone (Funakoshi), anti-single-stranded DNA (ssDNA; Shibayagi, Gunma, Japan), and anti-double-stranded DNA (dsDNA; Medical & Biological Laboratories) Ab and rheumatoid factor (Shibayagi). These ELISA plates were incubated with serum samples diluted 1:100. Relative levels of autoantibodies were determined for each group of mice using pooled serum samples. Sera were diluted at log intervals (1:10 to 1:105) and assessed for relative autoantibody levels as above except that the results were plotted as OD versus dilution (log scale). The dilutions of sera giving half-maximal OD values were determined by linear regression analysis, thus generating arbitrary units per ml values for comparison between sets of sera. To determine Ab concentrations in sera, ELISA was performed as described,20Engel P Zhou L-J Ord DC Sato S Koller B Tedder TF Abnormal B lymphocyte development, activation and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (490) Google Scholar using affinity-purified mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA (Southern Biotechnology Associates) to generate standard curves. The relative Ig concentration of each sample was calculated by comparing the mean OD obtained for duplicate wells to a semilog standard curve of titrated standard Ab using linear regression analysis. Total RNA was isolated from lower back skin with RNeasy spin columns (Qiagen, Crawley, UK). Total RNA from each sample was reverse-transcribed into cDNA. Expression of IL-4, IL-6, IL-10, IFN-γ, TGF-β1, TNF-α, and MIP-2 was analyzed using a real-time PCR quantification method according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequence-specific primers and probes were designed by Pre-Developed TaqMan assay reagents or Assay-On-Demand (Applied Biosystems). Real-time PCR (40 cycles of denaturing at 92°C for 15 seconds and annealing at 60°C for 60 seconds) was performed on an ABI Prism 7000 sequence detector (Applied Biosystems). Glyceraldehyde-3-phosphate was used to normalize mRNA. Relative expression of real-time PCR products was determined by using the ΔΔCt method22Meijerink J Mandigers C van de Locht L Tonnissen E Goodsaid F Raemaekers J A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.J Mol Diag. 2001; 3: 55-61Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar to compare target gene and housekeeping gene mRNA expression. One of the control samples was chosen as a calibrator sample. Splenic B cells were purified (>95% B220+) by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal, Lake Success, NY), and subsequently lysed in buffer containing 1% Nonidet P-40 as described.23Fujimoto M Poe JC Jansen PJ Sato S Tedder TF CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.J Immunol. 1999; 162: 7088-7094PubMed Google Scholar The purified splenic B cells were stimulated in 0.6 ml of culture medium in 48-well flat-bottom plates with 25, 50, or 100 ng/ml of BLM in the presence of 1 μg/ml of lipopolysaccharide (LPS, Sigma-Aldrich). In other experiments, B cells were stimulated with 200 μg/ml of hyaluronan that contained both high- and low-molecular weight hyaluronan (50 to 8000 kDa; Biomedicals, Irvine, CA), 200 μg/ml only low-molecular weight hyaluronan (15 to 40 kDa; R&D Systems, Minneapolis, MN), 100 μg/ml heparan sulfate (Biomedicals), 200 μg/ml chondroitin sulfate (Biomedicals), or 500 ng/ml high-mobility group box 1 protein (HMGB-1, Sigma-Aldrich) for 10 hours. Anti-mouse TLR4 mAb (Imgenex, San Diego, CA) or control rat IgG2a (R&D Systems) was added 60 minutes before hyaluronan stimulation at concentrations of 100 μg/ml. Expression of IL-4, IL-6, IL-10, IFN-γ, TGF-β1, TNF-α, and MIP-2 was analyzed using a real-time PCR quantification method. Culture supernatants from unstimulated or stimulated B cells were also analyzed for the production of these cytokines by specific ELISA kits. All data are expressed as mean values ± SD. The Mann-Whitney U-test was used to determine the level of significance of differences between sample means, and Bonferroni's test was used for multiple comparisons. BLM was injected subcutaneously into the backs of mice daily for 4 weeks. Previous studies have shown that skin fibrosis, lung fibrosis, epithelial injury, and inflammatory cell infiltration develop during the first 4 weeks of BLM treatment, peak in the 4th week, and begin to resolve 6 weeks after the cessation of treatment.18Yamamoto T Takagawa S Katayama I Yamazaki K Hamazaki Y Shinkai H Nishioka K Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma.J Invest Dermatol. 1999; 112: 456-462Crossref PubMed Scopus (339) Google Scholar, 24Yamamoto T Takahashi Y Takagawa S Katayama I Nishioka K Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice.J Rheumatol. 1999; 26: 2628-2634PubMed Google Scholar, 25Yamamoto T Kuroda M Nishioka K Animal model of sclerotic skin. III: Histopathological comparison of bleomycin-induced scleroderma in various mice strains.Arch Dermatol Res. 2000; 292: 535-541Crossref PubMed Scopus (80) Google Scholar, 26Nakao A Fujii M Matsumura R Kumano K Saito Y Miyazono K Iwamoto I Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.J Clin Invest. 1999; 104: 5-11Crossref PubMed Scopus (387) Google Scholar, 27Yaekashiwa M Nakayama S Ohnuma K Sakai T Abe T Satoh K Matsumoto K Nakamura T Takahashi T Nukiwa T Simultaneous or delayed administration of hepatocyte growth factor equally represses the fibrotic changes in murine lung injury induced by bleomycin. A morphologic study.Am J Respir Crit Care Med. 1997; 156: 1937-1944Crossref PubMed Scopus (201) Google Scholar In this study, skin and lung fibrosis in CD19−/− and WT mice treated with either BLM or PBS was histopathologically assessed 1, 2, 3, and 4 weeks after the initiation of BLM treatment. Dermal thickness and lung fibrosis score showed time-dependent increases in BLM-treated mice (Figure 1, a and d). After 2 weeks of treatment, BLM treatment induced significantly greater dermal thickness relative to PBS treatment in WT mice (P < 0.05) but not in CD19−/− mice, although there was no significant difference in the dermal thickness between WT and CD19−/− mice at this time point. After 3 weeks, a significant difference in the dermal thickness between WT and CD19−/− mice was apparent (P < 0.005). After 4 weeks, the dermal thickness in BLM-treated WT mice significantly increased by 1.9-fold compared with PBS-treated WT mice (P < 0.005; Figure 1, a–c). In contrast, BLM-treated CD19−/− mice showed moderate thickening of dermal tissue that was significantly 27% thinner than that found in BLM-treated WT mice (P < 0.005), but remained thicker than that of PBS-treated CD19−/− and WT mice (P < 0.005). Masson trichrome staining revealed thickened collagen bundles in the skin from BLM-treated WT mice, which was also reduced by CD19 deficiency (data not shown). Similar results were obtained for the lung fibrosis score, except that a significant difference in the lung fibrosis score between WT and CD19−/− mice was detected after 2 weeks (Figure 1d). After 4 weeks of BLM administration, BLM-treated WT mice exhibited extensive inflammatory infiltration, fibrosis, granulomas, and alveolar epithelial injury (Figure 1, e and f). In contrast, CD19 deficiency reduced such histological changes. Thus, subcutaneous BLM injection induced skin and lung fibrosis that CD19 deficiency attenuated. The numbers of mast cells, macrophages, T cells, and B cells have been reported to increase in sclerotic skin from human SSc.5Kalogerou A Gelou E Mountantonakis S Settas L Zafiriou E Sakkas L Early T cell activation in the skin from patients with systemic sclerosis.Ann Rheum Dis. 2005; 64: 1233-1235Crossref PubMed Scopus (102) Google Scholar, 25Yamamoto T Kuroda M Nishioka K Animal model of sclerotic skin. III: Histopathological comparison of bleomycin-induced scleroderma in various mice strains.Arch Dermatol Res. 2000; 292: 535-541Crossref PubMed Scopus (80) Google Scholar, 28Hawkins RA Claman HN Clark RA Steigerwald JC Increased dermal mast cell populations in progressive systemic sclerosis: a link in chronic fibrosis?.Ann Intern Med. 1985; 102: 182-186Crossref PubMed Scopus (220) Google Scholar, 29Whitfield ML Finlay DR Murray JI Troyanskaya OG Chi JT Pergamenschikov A McCalmont TH Brown PO Botstein D Connolly MK Systemic and cell type-specific gene expression patterns in scleroderma skin.Proc Natl Acad Sci USA. 2003; 100: 12319-12324Crossref PubMed Scopus (353) Google Scholar Therefore, we counted these immune cells at the BLM-injected sites. Among WT mice, the numbers of mast cells, macrophages, T cells, and B cells were greater in BLM-treated WT mice than in PBS-treated WT mice (P < 0.005; Figure 2, a–d, respectively). BLM-treated CD19−/− mice showed lower infiltration of these cells than BLM-treated WT mice (P < 0.005), but still greater infiltration than PBS-treated CD19−/− mice (P < 0.005). It has been suggested that IL-4, IL-6, IL-10, IFN-γ, TNF-α, and TGF-β1 production contributes to BLM-induced fibrosis by regulating the production of collagen and glycosaminoglycans by fibroblasts.18Yamamoto T Takagawa S Katayama I Yamazaki K Hamazaki Y Shin
Referência(s)